Our long term goal is to define the molecular mechanism of store-operated Ca2+ entry (SOCE) in vascular smooth muscle cells (SMC) and other cell types. Recently we identified Ca2+-independent phospholipase A2 (iPLA2) as a novel determinant of SOCE, which was successfully confirmed by others. We discovered a novel plasma membrane-delimited cascade of reactions involving CIF-induced displacement of inhibitory CaM from iPLA2 which leads to its activation and production of lysophospholipids that in turn activate SOC channels. The importance of iPLA2 was fully confirmed in a recent screen of Drosophila genes, in which iPLA2, STIM1 and Orai1 (but not TRP) were found to play equally important roles in SOCE, but the mechanisms of their molecular and functional interactions are yet to be determined. Our preliminary studies strongly suggest that iPLA2 may be a key intermediate between STIM1 and Orai1. Based on the careful analysis of the most recent discoveries in the field, critical evaluation of different models, and our extensive preliminary findings, we propose a novel model of SOCE complex (SOCEplex) that brings a new dimension to SOCE mechanism, unifies conformational coupling and diffusible messenger ideas, involves STIM1, CIF, iPLA2 and Orai1, and offers their new structural and functional arrangement. The main hypothesis of this proposal is that iPLA2 is a central multifunctional element of the SOCEplex, and serves as a plasma membrane anchor for endoplasmic reticulum STIM1, acceptor of the signal from depleted stores and transducer of the signal to Orai1. Novel model of SOCE will be tested in primary vascular SMC and model cell lines using our integrative approach, which involves advanced molecular, biochemical, imaging, electrophysiological and functional methods. All approaches and methods have been established and successfully used in the PI's lab. The feasibility of our new hypothesis and success of the proposed studies are fully supported by extensive preliminary data and expertise of the PI and collaborators. Specific Aims of this proposal are: Aim 1: To establish iPLA2 as a molecular partner of STIM1 and Orai1, and a keystone of SOCEplex. We will assess alternative models of SOCE, establish a new role of iPLA22 as a linker between STIM1 and Orai1, identify the molecular mechanism of iPLA2 interaction with STIM1 and its role in SOCE, determine the mechanism of iPLA2 oligomerization and identify molecular domains in iPLA2 that are crucial for signal transduction in SOCEplex. Aim 2: To establish the roles of CIF and LysoPL in signal transduction within SOCEplex. We will confirm the role of CIF in signal transduction from depleted stores to plasma membrane, determine the role of lipid rafts, and assess LysoPL-mediated mechanism of Orai1 activation. PUBLIC HEALTH RELEVANCE: Our long term goal is to define the mechanism of store-operated Ca2+ entry (SOCE) pathway that is crucial for a wide variety of physiological functions. The goals of this proposal are to establish the role of specific plasma membrane-bound isoform of Ca2+-independent phospholipase A2 as a crucial component of SOCE machinery, and to demonstrate that it serves as a central molecular, structural and functional element of SOCE complex (SOCEplex) that transduces the signal from depleted stores to plasma membrane channels. The feasibility of our novel hypothesis is fully supported by extensive preliminary data and advanced expertise of PI's lab.